Ubiquitination primarily serves as a targeting signal, and proteins carrying the most common type of poly-Ubiquitin chain are targeted for destruction by the ubiquitin-proteasome pathway, responsible for the majority of cytosolic proteolysis (Ciechanover et al., Proc. Natl. Acad. Sci. USA, 95, 2727-30, 1998), Ubiquitin (Ub) is attached to proteins through an isopeptide linkage, involving the C-terminal carboxylate of Ub and the c-NH2 of a lysine side chain, (Ciechanover et al., Mot Biol Rep, 26, 59-64, 1999; Hodgins et al., J. Biol. Chem., 271, 30 28766-28771, 1996). The enzyme cascade involved in Ub-conjugation and poly-Ub chain formation comprises at least three distinct sets of enzymatic activities including the Ub activating enzyme E1, Ub-conjugating enzymes (E2) and E3 ligases (reviewed in Hershko and Ciechanover, Annu Rev Biochem, 67, 425-79, 1998),
Removal of Ub is carried out by deubiquitinating enzymes (DUBs) or deconjugating enzymes (DCEs). These are a large family of proteases that can release poly-Ub chains from proteins to be degraded by the 26S proteasome, recycle monomeric Ub, liberate Ub from the Ub-fusion protein precursors, reverse regulatory ubiquitination and edit inappropriately ubiquitinated proteins (reviewed in Chung et al., Biochem Biophys Res Comm, 266, 633-40, 1999). DUBs can be subdivided into Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs). In vitro, UBPs hydrolyze isopeptide bonds between Ub and folded protein domains, such as additional Ub moieties or target proteins. Thus, UBPs exhibit broad substrate specificity (Wilkinson, FASEBJ, 11, 1245-56, 1997). UCHs generally cleave bonds between Ub and an unfolded polypeptide or Ub and small substituents (Pickart et al., J. Biol. Chem., 260, 7903-10, 1985; Wilkinson, FASEB J, 11, 1245-56, 1997; Wilkinson et al., Biochemistry, 25, 6644-9, 1986). Deletion studies in yeast suggest that the: substrate specificities of UCHs and UBPs overlap (Amerik et al., Biol Chem, 381, 981-92, 15 2000; Baker et al., J Biol Chem, 267, 23364-75, 1992). Both UBPs and UCHs can associate with the 26S proteasome and are involved in the regulation of Ub-dependent proteolysis: (Voges et al., Annul Rev. Biochem, 68, 1999).
Ubiquitination and deubiquitination are emerging as regulatory mechanisms controlling, e.g., proteolysis, protein-protein interactions, DNA repair, and cellular signaling. Recently, USP2 and UCH37 have been shown to deubiquinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy, e.g., for treating cancer. The broad involvement of ubiquitin systems in cellular processes, including proliferation of cancer cells, provides an attractive set of potential drug targets. Most assay formats rely heavily on low throughput methods or customized reagents.
Small Ubiquitin-related Modifier (SUMO) proteins are small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. SUMO proteins are similar to ubiquitin (Ulrich, Trends Cell Biol. 2005 Oct.; 15(10):525-32). In contrast to ubiquitin, SUMO is typically not used to tag proteins for degradation. The protein is typically not active until the last four amino acids of the C-terminus have been cleaved off.
The majority of non-radioactive kinase assays depend on phosphorylation of a chemically synthesized peptide substrate of up to approximately 20 residues. Although, it would be preferable to use larger native substrates (such as whole proteins or protein domains) that are “physiologically relevant” (i.e. they can be the “native” substrate of a kinase in a biologically relevant pathway). The use of “native” substrates is a desirable feature for many practitioners of kinase assays. As an example, it is known that some kinases require a “docking” site far removed from the site of phosphorylation in order to be phosphorylated. In some cases, smaller peptide substrates do not function as substrates.
Drug discovery can involve the systematic and/or high-throughput screening of diverse chemical libraries containing thousands of members. The size and complexity of these libraries, when coupled with the expense and length of the FDA approval process, have resulted in the need for simple, efficient, and homogeneous assays for probing molecular interactions.
Luminescence-based techniques, including fluorescence polarization (FP), resonance energy transfer (RET), and luminescence resonance energy transfer methods (LRET) methods, are typically highly sensitive, homogenous methods for probing molecular interactions. Background luminescence (e.g., fluorescence or luminescence from assay components) and non-specific interactions of assay components, however, can limit the sensitivity of luminescence-based assays, particularly when luminophores having short lifetimes are used, resulting in the detection of false positives or false negatives in a drug or compound screen. Follow-up screening of individually-picked compounds or the use of multiple screens may be required to validate screen results. It would be useful to have screening methodologies that could increase the information content of fluorescent or luminescent assays and reduce the number of spurious results encountered in drug screens.